Myelin restoration by antisense modulation of HuR and HuD ELAV RNA binding protein expression in mouse models of multiple sclerosis

doi:10.21203/rs.3.rs-1858703/v1

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Myelin restoration by antisense modulation of HuR and HuD ELAV RNA binding protein expression in mouse models of multiple sclerosis

https://doi.org/10.21203/rs.3.rs-1858703/v1

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published 25 Jan, 2023

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Neuropathic pain is the most difficult to treat pain syndrome in multiple sclerosis. Evidence relates neuropathic pain to demyelination that often originates from unresolved neuroinflammation or altered immune response. Post–transcriptional regulation of gene expression might play a fundamental role in the regulation of these processes and the aim of this study was to investigate ELAV RNA binding proteins in demyelination-associated neuropathic pain states. The ubiquitous HuR and the neuronal HuD are involved in inflammatory phenomena and neuronal development and maintenance, respectively. Thus, we investigated their role in the mouse experimental autoimmune encephalomyelitis (EAE) model. HuR resulted overexpressed in the spinal cord of MOG–EAE and PLP–EAE mice and was detected in CD11b+ cells. Intranasal delivery of an antisense oligonucleotide (ASO) to knockdown HuR increased myelin basic protein expression and Luxol Fast Blue staining, indication of increased myelin content, in both EAE models. These effects temporally coincided with attenuation of pain hypersensitivity. Conversely to HuR, HuD was largely downregulated in the MOG-EAE spinal cord, along with GAP43 and Neurofilament H, while in PLP-EAE mice HuD and neuronal markers remained unaltered. In MOG-EAE mice anti–HuR ASO increased the expression of HuD in GAP43 expressing cells and promoted a HuD–mediated neuroprotective activity. In PLP-EAE mice HuR silencing dampened pro–inflammatory response mediated by spinal microglia activation. In conclusion, anti–HuR ASO showed myelin protection at analgesic doses with multitarget mechanisms, and it deserves further consideration as an innovative agent to counteract demyelination in neuropathic pain states.

HuR

HuD

ELAV RNA binding protein

neuropathic pain

myelin

microglia

experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) is a chronic inflammatory condition of the central nervous system (CNS) characterized by demyelination with concomitant axonal and neuronal degeneration [1]. Among the symptoms and signs of MS patients, pain affects about the 70% of patients and neuropathic pain represents the most difficult to treat chronic pain syndrome [2]. A complete and effective relief from neuropathic pain is usually not achieved and available treatments produce limited efficacy in a small portion (20–40%) of patients. In fact, therapy is often unsatisfactory due to poor efficacy and appearance of unwanted side effects [3, 4]. Treatments for neuropathic pain do not focus on resolving the cause of the pain, but rather on merely suppressing symptoms. Therefore, understanding the causal processes of neuropathic pain and their modulation could constitute an effective therapeutic strategy.

Nerve injury induces a pro–inflammatory response in the spinal cord in rodents and humans and can cause neuropathic pain [5, 6]. Spinal microglia have traditionally been implicated in this pro–inflammatory response and in the promotion of pain hypersensitivity [7, 8]. However, anti–inflammatory treatments did not relieve pain in patients with neuropathies [9, 10], raising the possibility that additional cellular mechanisms or interactions might influence neuroimmune responses and pain hypersensitivity in neuropathic animals.

Recent evidence suggests that neuropathic pain is related to demyelination, which is the loss or damage of the myelin sheath that lines the nerve fibres, causing impaired nerve transmission [11]. Frequently at the basis of demyelination there is a not resolved chronic inflammation process [12]. Microglia can communicate with neurons through the release of several mediators, such as growth factors, cytokines, neurotrophins, or through direct contact [13, 14], making glia–neuron interaction a novel target for novel therapies for demyelinating diseases [15].

Post–transcriptional processing of gene expression and RNA stabilization of inflammatory proteins play a fundamental role in the regulation of a pro–inflammatory response. The Embryonic lethal abnormal vision (ELAV)/Hu proteins represent a family of RNA binding proteins (RBPs) that play a key role in the fate of new transcribed mRNA. There are four mammalian ELAV RBPs encoded by separate genes: HuB, HuC, HuD (neuron–specific), and HuR (ubiquitously expressed) [16]. HuR is expressed in activated microglia [17, 18] and it is involved in cytoplasmic stabilization and/or enhancement of translation of mRNA of pro–inflammatory mediators, representing an important regulator of inflammatory and immune processes [19]. Neuronal ELAVs (nELAVs) accelerate neuronal differentiation and axonal outgrowth, and modulate neuronal integrity [20]. An interplay between nELAV and HuR has been reported [21], indicating that these proteins act in concert. Thus, HuR overexpression could promote inflammatory phenomena, which might lead to neurotoxic consequences. In addition, an aberrant HuR content might imbalance nELAV/HuR protein ratio promoting demyelination and neurodegeneration.

All this evidence makes HuR and HuD potential targets to attenuate demyelination in neuropathic pain states. Verification of this hypothesis has been pursued in mouse models of MS (Experimental Autoimmune Encephalomyelitis, EAE) through an antisense oligonucleotide (ASO)–based approach to control HuR–mediated and HuD–mediated post–transcriptional regulation of gene expression.

Animals

Experiments for SNI were performed on male CD1 mice (24–26 g, 4–week–old), for MOG₃₅₋₅₅ EAE model were performed on CB57BL/6 female mice (20–25 g, 10–week–old) and for PLP_139–151EAE were performed on SJL female mice (18–20 g, 10–week–old) from Envigo (Varese, Italy). Animals were housed in the Ce.S.A.L. (Centro Stabulazione Animali da Laboratorio, University of Florence) vivarium and used one day after their arrival. Mice were housed in standard cages, kept at 23 ± 1 °C with a 12–h light/dark cycle, light on at 7 a.m., and fed with standard laboratory diet and tap water ad libitum. The cages were placed in the experimental room 24 h before behavioral testing for acclimatization and all tests were conducted during the light phase.

Mice were sacrificed by cervical dislocation for removal of spinal cord for in vitro analyses. The number of animals per experiment was based on a power analysis [22]. For behavioral assays all tested groups comprised eight animals. For in vitro assays, data are mean of five individual experiments.

Antisense oligonucleotide administration

Phosphorothioate oligonucleotides (ODNs) (resistant to exonuclease–mediated degradation) were used (Tib Molbiol, Genoa, Italy). The antisense ODN (ASO) against HuR was the following: 5’–A*T*AACCATTAGACATT*G*T–3, where the asterisks indicate the phosphorothioate phosphate groups. The ASO against HuD was the following: 5′–G*T*TCTGGAGCCTCATC*T*T–3′ where the asterisks indicate the phosphorotioate phosphate groups. A 18–mer fully degenerate ODN (dODN), where each base was randomly G, or C, or A, or T, was used as control ODN treatment. To enhance both uptake and stability, ODNs were preincubated at 37 ̊C for 30 minutes with 13 µM DOTAP (Sigma, Milan, Italy), an artificial cationic lipid. ODNs were administered at the dose of 1 nmol/mouse on the basis of previous results [23, 24]. The experimental protocol to test the effect of ASO on behavioral and in vitro tests included 3 control groups: dODN (as control ODN), vehicle (DOTAP 13 µM), and saline.

Intranasal administration

For intranasal (i.n.) administration, mice were slightly anesthetized by 2% isoflurane inhalation and placed in a supine position. A 5–µL aliquot of DOTAP, dODN, or ASO was slowly dropped alternatively to each nostril with a micropipette tip. A total of 10 µL of solution containing 1 nmol of ODNs was delivered to mice. In the EAE model, treatments performed before immunization might neutralize the effect of an artificial disease induction and do not always address the therapeutic potential of drugs for patients at risk. HuR or HuD silencing was, thus, produced starting from day 14 from immunization, corresponding to the first disease peak. To achieve the protein knockdown, mice received a single i.n. injection every 2 days. The spinal cords were removed on day 30 post–immunization. In the SNI model treatments were delivered every 2 days starting from day 3 post-surgery and the spinal cords for in vitro tests were removed on day 10.

EAE induction

Sex–related differences of innate and adaptive immune responses may account for a prevalent female susceptibility to develop EAE symptoms [25]. Myelin Oligodendrocyte Glycoprotein (MOG_35–55) was used to induce chronic progressive EAE in CB57BL/6 female mice [26] and myelin proteolipid protein (PLP_139–151) was used to induce relapsing–remitting EAE in SJL female mice [27]. Animals were immunized subcutaneously (s.c.) in the flanks and at the base of the tail with a total of 200 µg of PLP_139–151or MOG_35–55 peptide (synthesized by EspiKem Srl., University of Florence, Italy) per animal emulsified in complete Freund adjuvant (CFA; Sigma, Milan, Italy) supplemented with 4 mg/ml of Mycobacterium tuberculosis (strain H37Ra; Difco Laboratories, Detroit, Michigan, USA). Control mice received CFA without antigen. Immediately thereafter, and again 48 h later, all mice received an intraperitoneal (i.p.) injection of 500 ng Pertussis Toxin (Sigma, Milan, Italy) in 100 µl phosphate buffer saline (PBS). General health and body weights of all mice were assessed prior to immunization and once–daily thereafter in a blinded manner until the completion of study. Locomotor coordination and nociceptive threshold were analyzed before onset and regularly during the course of the disease.

Spared nerve injury (SNI)

Behavioral testing was performed before surgery to establish a baseline for comparison with postsurgical values. Mononeuropathy was induced by SNI as previously described [28]. Mice were anesthetized with a mixture of 4% isoflurane in O₂/N₂O (30:70 v/v) and placed in a prone position. The right hind limb was slightly elevated, and a skin incision was made on the lateral surface of the thigh. The sciatic nerve was exposed at mid–thigh level distal to the trifurcation and freed of connective tissue; the three peripheral branches (sural, common peroneal and tibial nerves) of the sciatic nerve were exposed without nerve structures. Both tibial and common peroneal nerves were ligated with a microsurgical forceps (5.0 silk, Ethicon; Johnson & Johnson Intl, Brussels, Belgium) and transected together. The sural nerve was carefully preserved by avoiding any nerve stretch or contact with surgical tools. Muscle and skin were closed in two distinct layers with silk 5.0 sutures. Intense, reproducible, and long–lasting thermal and mechanical allodynia–like behaviors are measurable in the non–injured sural nerve skin extensions. The sham procedure consisted of the same surgery without ligation and transection of the nerves.

Behavioral testing

Animals were habituated to the testing environment daily for at least 2 days before baseline testing. To evaluate onset and progression of pain hypersensitivity, neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. Nociceptive responses to mechanical and thermal stimulus were measured every 30 min for 3 h before and after nerve surgery. Each mouse served as its own control, the responses being measured both before and after surgery. EAE mice were monitored 3, 7, 10, 14, 18, 20, 25, 28 and 30 days after immunization. All testing was performed with a blind procedure.

Von Frey test Mechanical allodynia was measured by using Dynamic Plantar Aesthesiometer (Ugo Basile, Bologna, Italy), as described [24]. The mice were placed in individual Plexiglas cubicles (8.5 cm L 3,4 cm H 3,4 cm) on a wire mesh platform and allowed to acclimate for approximately 1 h, during which exploratory and grooming activity ended. After that, the mechanical stimulus was delivered to the plantar surface of the hind paw of the mouse from below the floor of the test chamber by an automated testing device. A steel rod (2 mm) was pushed with electronic ascending force (0–5 g in 35 s). When the animal withdrew its hind paw, the mechanical stimulus was automatically withdrawn, and the force recorded to the nearest 0.1 g. Nociceptive response for mechanical sensitivity was expressed as mechanical paw withdrawal threshold (PWT) in grams. PWT was quantified by an observer blinded to the treatment.

The mean PWT was calculated from six consecutive trials (each performed every 30 min) and averaged for each group of mice.

Hargreaves’ plantar test Thermal nociceptive threshold was measured using Hargreaves’ device, as described [29]. Paw withdrawal latency in response to radiant heat (infrared) was assessed using the plantar test apparatus (Ugo Basile, Comerio, Italy). Each mouse was placed under a transparent Plexiglas box (7.0 × 12.5 cm², 17.0 cm high) on a 0.6–cm–thick glass plate and allowed to acclimatize for 1–2 h before recording. The radiant heat source consisted of an infrared bulb (Osram halogen–bellaphot bulb; 8 V, 50 W) that was positioned 0.5 cm under the glass plate directly beneath the hind paw. The time elapsed between switching on the infrared radiant heat stimulus and manifestation of the paw withdrawal response was measured automatically. The intensity of the infrared light beam was chosen to give baseline latencies of 10 s in control mice. A cut–off of 20 s was used to prevent tissue damage. Each hindpaw was tested 2–3 times, alternating between paws with an interval of at least 1 min between tests. The interval between two trials on the same paw was of at least 5 min. Nociceptive response for thermal sensitivity was expressed as thermal paw withdrawal latency in seconds. All determinations were averaged for each animal.

Clinical Disease Score Clinical disease scoring of EAE and sham mice (control group) was undertaken once daily in a blinded manner to evaluate the severity and extent of motor function deficits using a 5–point scale with half–point gradations [30]. EAE scores were daily assessed: score 0, no obvious changes in motor functions; score 0.5, distal paralysis of the tail; score 1, complete tail paralysis; score 1.5, mild paresis of 1 or both hind legs; score 2, severe paresis of hind legs; score 2.5, complete paralysis of 1 hind leg; score 3, complete paralysis of both hind legs; and score 3.5, complete paralysis of hind legs and paresis of 1 front leg. EAE clinical disease was classified as present by clinical scores ≥ 1, whereas clinical scores ≤ 0.5 were regarded as disease remission or absence. Mice reaching a score of 3.5 were excluded from the study.

Rotarod Test The apparatus consisted of a base platform and a rotating rod with a diameter of 3 cm and a non–slippery surface. The rod was placed at a height of 15 cm from the base. The rod, 30 cm in length, was divided into 5 equal sections by 6 disks. Thus, up to 5 mice were tested simultaneously on the apparatus, with a rod–rotating speed of 16 r.p.m. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 30 s, as described [31]. The performance time was measured before and regularly after immunization.

Western blot analysis

Samples were homogenized in a homogenization buffer containing 25 mM Tris–HCl pH=7.5, 25 mM NaCl, 5 mM EGTA, 2.5 mM EDTA, 2 mM sodium pyrophosphate (NaPP), 4 mM p–nitrophenylphosphate (PNFF), 1 mM Na₃VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, 50 µg/ml aprotinin, 0.1% SDS. The homogenate was centrifuged at 9,000 x g for 15 min at 4°C, the low–speed pellet was discarded. Protein concentration in the supernatant (whole cell lysate) was quantified using the Bradford’s method (protein assay kit, Bio–Rad Laboratories, Milan, Italy). Membrane homogenates (20–50 mg) were separated on 10% SDS–PAGE and transferred onto nitrocellulose membranes (120 minutes at 100 V) using standard procedures. Blots were incubated overnight at 4 ̊C with specific antibodies against IBA1 (1:1000; sc–32725; RRID:AB_667733), HuD (1: 1000; sc–48421; RRID:AB_627766); HuR (1:1000, sc–5261; RRID:AB_627770), MBP (1:1000 sc–271524; RRID:AB_10655672), S100 α/ß (1:500,sc–58839; RRID:AB_2183338) GAP43 (1:500 sc–17790; RRID:AB_627660) (Santa Cruz Biotechnology Inc, Santa Cruz, CA), Neurofilament H (1:1000 bs–0708R; RRID:AB_10855865) (Bioss, Boston, MA). Blot visualization (chemiluminescence detection system) and signal intensity quantification (ImageJ, Wayne Rasband, National Institute of Health, USA) were performed. The exposition and developing time used was standardized for all the blots. Several reports suggest that commonly used housekeeping proteins are not equally expressed across cell types and experimental conditions and quantification normalization of signal intensity to total protein loading is preferred [32]. For each sample, the signal intensity was normalized to that of total protein stained by Ponceau S and the acquired images were quantified using Image Lab software. Measurements in control samples expressed as fold to control value to control for unwanted sources of variation.

Immunofluorescence

For immunofluorescence analysis biological samples have been fixed in formalin at 4% for 24 h, dehydrated in alcohol, included in paraffin, and finally cut into 20 μm sections (spinal cords) and 10 μm (sciatic nerve, dorsal root ganglia). Primary antibodies used were antibodies for microglia (CD11b) (1:100; bs–11127; RRID:AB_10856024), Neurofilament H (1:100 bs–0708R; RRID:AB_10855865) (Bioss, Boston, MA), HuD (1: 100; sc–48421; RRID:AB_627766); HuR (1:100, sc–5261; RRID:AB_627770), MBP (1:100, sc–271524; RRID:AB_10655672), S100 α/ß (1:100, sc–58839; RRID:AB_2183338) and GAP43 (1:500 sc–17790; RRID:AB_627660). After rinsing in PBS containing 0.01% Triton–X–100, sections were incubated in secondary antibodies labelled with Invitrogen Alexa Fluor 488 (490–525, 1:400; RRID:AB_221544), Invitrogen Alexa Fluor 568 (578–603, 1:400; RRID:AB_2534072) (Thermo Fisher Scientific), and Cruz Fluor 594 (592–614, 1:400; RRID:AB_2847914) (Santa Cruz Biotechnology) at room temperature for 2 hours. Sections were coverslipped using Vectorshield mounting medium (Vector Laboratories, Burlingame, CA). A Leica DM6000B fluorescence microscope equipped with a DFC350FX digital camera with appropriate excitation and emission filters for each fluorophore was used to acquire representative images. Images were acquired with 35 to 340 objectives using a digital camera. The immunofluorescence intensity was calculated using Image J.

Evaluation of BBB disruption

Under normal conditions Evans Blue (EB) dye conjugates with serum albumin to form a large complex that is not able to cross an intact barrier. However, when BBB is disrupted, this complex can entry the CNS [33]. Thus, the level of BBB disruption was detected by quantitative measurement for Evans blue content, as previously described [18] at day 28 after immunization. Briefly, mice were intraperitoneally injected with 2% Evans blue solution at a dose of 5.0 ml/kg per mouse. The dye was allowed to circulate for 4 h, and the mice were subsequently anesthetized and perfused transcardially with saline to remove the Evans blue dye in the vascular system. The brain and lumbar spinal cord were immediately removed, and images were captured. Tissues were homogenized with 2.5 ml PBS and mixed with 2.5 ml 50% trichloroacetic acid to precipitate protein overnight at 4 °C. The samples were centrifuged for 30 min at 10,000 × g, and the supernatants were measured at 610 nm for the absorbance of Evans blue by using a MP96 spectrophotometer (Safas, Monaco). The Evans blue content was expressed as micrograms per gram of brain and lumbar spinal cord.

Luxol fast blue (LFB) myelin staining

LFB is a commonly used method to visualize myelin through an optical microscope and it is used to detect demyelination in the central nervous system. Spinal cord samples have been fixed in formalin at 4% for 24 h, dehydrated in alcohol, included in paraffin, and finally cut into 20 μm sections and placed on a glass slide. Then, slides have been washed 2 times in PBSB–Tryton 0.3% and left to complete drying. After that, colorant LFB has been added on the slides and put at 60° C for 4 hours. Subsequently, slides have been washed in EtOH 96% and in H₂O. The decolorization step has been made with lithium carbonate (0.1% in H₂0) for 15 seconds and EtOH 70% for 30 seconds. The decolorization step has been made until there was a contrast between the blue of the white and the grey matter. Afterwards, final washings have been made with: EtOH 96% for 3 minutes, EtOH 100% for 3 minutes, Xylene for 5 minutes. Finally, we mounted the slides with a mounting medium and stored at room temperature. The slides were viewed on an OLYMPUS BX63F.

Determination of TNF–α, IL–1β, IL–6 and IL–17 from plasma

Mice were put under general anesthesia by intraperitoneal injection of zoletil and xylazine cocktail. Blood samples were taken from the ventricle with a standard cardiac puncture method, centrifuged at 3000 × g for 10 min at 4 °C, and the plasma was collected. TNF–α, IL–1β, and IL–17 protein levels were evaluated by using non–competitive sandwich ELISA (Biolegend e–Bioscience DX Diagnostic, Monza, Italy), following the supplier instructions. The absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monaco), and cytokine levels were expressed as picograms per milliliter according to a standard calibration curve.

Statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology [34]. Results are expressed as mean ± SEM. Repeated measures two–way analysis of variance (ANOVA) followed by the Bonferroni test was used to compare locomotor behavior, pain behaviors between neuropathic and sham–mice. The Kruskal–Wallis non–parametric test followed by Dunn's test was used to compare score values between EAE–mice administered ASO, dODN or vehicle. Score values comparison between EAE– and control mice was assessed using the non–parametric Wilcoxon test. One–way ANOVA followed by the Tukey post–hoc test was used to compare remaining data. Sample sizes subjected to statistical analysis had at least five samples per group (n = 5), where n is equal to the number or independent values. The statistical significance criterion was P < 0.05. The computer program GraphPad Prism version 9.0 (GraphPad Software Inc, San Diego, CA) was used.

Anti–HuR ASO increased myelin, Neurofilament H and GAP43 in spinal cord of MOG–EAE mice

To evaluate the role of HuR and HuD in neuropathic pain associated with demyelination, we used the Experimental Autoimmune Encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The most widely used EAE models are the relapsing–remitting PLP_139–151–induced and the chronic MOG_35–55–induced models [35]. Both models are characterized by central neuropathic pain, extensive inflammation in the white matter, demyelination, whereas in the chronic MOG–model axonal degeneration was more prevalent [36].

The involvement of HuR in neuropathic pain related spinal demyelination was evaluated by protein knockdown through an anti–HuR ASO. The presence of an interplay between HuR and nELAVs was investigated by comparing the effect produced by anti–HuR ASO with those produced by anti–HuD ASO. We have recently demonstrated that anti–HuR ASO induced a comparable spinal HuR protein knockdown to i.t. administration [23]. On these bases, the ASOs were i.n. administered and their efficacy within the spinal cord was confirmed by western blotting experiments showing the HuR and HuD protein reduction following corresponding ASO administration (Fig. 1A). Degenerate ODN (dODN) was used as control ODN. No difference was observed among naïve, vehicle– and dODN–treated groups.

The level of myelination within the central nervous system (CNS) was detected in spinal cord samples of EAE mice by Luxol Fast Blue (LFB) staining. Representative images (Fig. 1B) and quantitative analysis (Fig. 1C) of spinal cord sections showed a robust myelin loss in MOG–EAE mice that was significantly increased by anti–HuR ASO. These results were consistent with our previous data obtained in PLP–EAE mice [37]. In spinal cord tissue of MOG–mice, a drastic reduction of MPB was observed that was markedly up–regulated by anti–HuR ASO (Fig. 1C). Anti–HuD ASO had no effect on either LFB staining or MBP expression. HuR is expressed on OPCs, as showed by colocalization with NG2, a marker of OPC. However, the anti–HuR ASO effect on myelination appeared not related to the promotion of OPC production since ASO did not alter expression of both OPCs (NG2) and progenitor cells (nestin) content in spinal cord samples (Supplementary Figure 1).

Neurodegeneration and axonal loss are prominent feature in MOG–EAE mice as demonstrated by the robust reduction of GAP43 (regeneration marker; Fig. 1E), and Neurofilament H (axonal marker; Fig. 1F) protein level. Silencing of HuR increased the levels of neuro–regeneration markers up to baseline control values whereas anti–HuD ASO didn’t provoke any effect on all markers evaluated.

Results from MOG–EAE were compared to those obtained from PLP–EAE mice, PLP–EAE mice, a model of relapsing–remitting MS mainly characterized by spinal neuroinflammation and a lower degree of axonal loss, that showed no significant modification of MBP (Fig. 1G), GAP43 (Fig. 1H) and neurofilament H (Fig. 1I) protein expression. Silencing of HuR produced a modest increase of neurofilament H expression and left unchanged the other two targets. Consistent with MOG–EAE data, anti–HuD ASO didn’t modify the expression of any markers evaluated.

ELAV silencing inhibits BBB permeability in EAE mice

One of the major features of EAE and MS is the dysfunction of the blood brain barrier (BBB). Endothelial cells express IL–17 receptor, and IL–17 destroys the tight junctions of the blood brain barrier (BBB) to facilitate the migration of CD4+ T cells in humans [38] and mice [39]. HuR can directly bind to the IL–17 mRNA, thus regulating IL–17 expression [40] and previous results showed the inhibition of BBB permeability by i.t. anti–HuR ASO in PLP–EAE mice [18]. We, thus, investigated the effect of i.n. anti–HuR and anti–HuD ASOs on BBB leakage. Quantification analysis showed an increase of dye extravasation in brain (Fig. 2A) and spinal cord (Fig. 2B) of MOG mice on day 28 post–immunization. Silencing of HuR largely decreased the EB content in both brain and spinal cord, returning to values comparable with the baseline level of the control mice. Anti–HuD reduced the dye content in the brain with an effect comparable to anti–HuR ASO. Conversely, in the spinal cord tissue there was a trend toward a BBB permeability reduction without reaching the statistical significance. Representative images of the brain and spinal cord showed that the degree of EB leakage (white intensity) was increased in EAE mice.

Effect of anti–HuR and anti–HuD ASOs on inflammatory mediators in EAE mice

Quantification of the plasma level of cytokines showed an increase of IL–17 (Fig. 2C), TNF–⍺ (Fig. 2E) and IL–6 (Fig. 2F) in both MOG and PLP mice. IL–1β plasma levels were significantly increased only in the PLP–treated group (Fig. 2D). Treatment with anti–HuR ASO educed the expression of IL–17, IL–1ß and IL–6 in PLP–mice with no effect on TNF–⍺ expression. No variation on circulating cytokine levels was observed in anti–HuR ASO treated MOG–EAE mice. Anti–HuD ASO never modified the increased levels of circulating cytokines (Fig. 2G).

Although the broad range of neurological symptoms and stages of disease progression, a key hallmark of EAE and MS is the neuroinflammation. In spinal cord tissue of MOG–EAE mice (Fig. 2H) and, more abundantly, of PLP–EAE mice (Fig. 2I), an increased expression of the microglia marker IBA–1 has been observed. IBA1 expression was reduced by anti–HuR ASO treatment in the PLP group while it remained unmodified in MOG mice. To confirm these data, immunofluorescence experiments (Fig. 2J) and quantification analysis (Fig. 2K) showed a modest increased expression of CD11b protein (a marker of activated state of microglia) in MOG–EAE mice that was not modified by HuR silencing. Results were compared to those obtained from PLP mice that showed a much more intense increase of CD11b expression that was abolished to baseline levels by anti–HuR ASO.

Effect of ELAV–silencing on EAE behavioral phenotype

MOG–EAE mice showed a significant reduction of the mechanical (Fig. 3A,F) and thermal threshold (Fig. 3B,G) in the hind paws from day 7 and day 14 post–immunization, respectively, that persisted until day 28. Consistent with clinical symptoms in MS patients, EAE mice also showed motor disability, as indicated by the progressive increase of clinical score (Fig. 3C,H), and locomotor disfunction, evaluated as impaired rotarod performance (Fig. 3D,I), from day 14 post–immunization up to day 28. EAE mice from day 14 showed a trend to a body weight loss compared with sham mice in which a constant and progressive increase of weight was recorded (Fig. 3EJ). Silencing of HuR reduced mechanical allodynia on day 28 (Fig. 3A) without affecting thermal hyperalgesia (Fig. 3B). Anti–HuR ASO attenuated rotarod impairment (Fig. 3D), with no significant effect on clinical score (Fig. 3C), and body weight loss (Fig. 3E).

HuD silencing improved motor dysfunction (Fig. 3I) with no effect on clinical score (Fig. 3H) and showed a trend toward a reduction of body weight loss (Fig. 3J), but, conversely to anti–HuR ASO, HuD ASO did not modify pain hypersensitivity (Fig. 3F,G),

To better define the role of HuR and HuD in central neuropathic pain, results from MOG–EAE mice were compared with results from PLP–EAE mice recorded at 28–day post–immunization. Consistently with MOG–EAE results, anti–HuR ASO attenuated mechanical allodynia (Fig. 3K) and thermal hyperalgesia (Fig. 3L) whereas anti–HuD AS was ineffective on pain hypersensitivity (Fig. 3K,L). Furthermore, anti–HuR ASO improved both clinical score and locomotor disability (Fig. 3M,N) in PLP–EAE model, resulting more effective in attenuating the overall EAE symptoms compared with the MOG–EAE model. These results are in line with the clinical efficacy of current therapies for MS in which a decrease of the relapse rate can be produced in relapsing–remitting patients, while efficacy in progressive forms is limited [41, 42]. In PLP–EAE mice anti–HuD ASO attenuated locomotor dysfunction (Fig. 3N), but it was ineffective on clinical score (Fig. 3M), and further aggravated body weight loss (Fig. 3O).

Effect of ELAV silencing on thermal and mechanical allodynia in peripheral neuropathic pain

To better define the role of ELAV isoforms in the central events involved in neuropathic pain associated with demyelination, we investigated the effects produced by i.n. ELAV silencing in a model of peripheral mononeuropathy (SNI) by comparing the nociceptive phenotype with modulation of main markers at central (spinal cord) and peripheral (sciatic nerve) level. In the ipsilateral side of spinal cord tissue of SNI mice, an increased immunostaining of HuR (Fig. 4A, red, upper panel) and HuD (Fig. 4A, green, upper panel) was detected in comparison with the contralateral side. These results have been confirmed with western blotting analysis (Fig. 4B). Silencing of HuR or HuD by i.n. administration of anti–HuR ASO (Fig. 4A, red, bottom) and anti–HuD ASO (Fig. 4A, green, bottom), respectively, reduced the corresponding protein expression.

Behavioral nociceptive tests showed that anti–HuR ASO reduced thermal (Fig. 4D) and mechanical (Fig. 4E) allodynia in SNI mice starting from 7 days after surgery whereas that anti–HuD ASO reduced pain hypersensitivity from post–surgical day 10. Both of ASOs didn’t affect mechanical threshold in the contralateral side (Fig. 4F).

Immunofluorescence experiments showed the colocalization of HuR with CD11b, a marker of activated state of microglia (Fig. 4G) whereas the neuronal ELAV HuD colocalized with Neurofilament H (NF–H) (Fig. 4H), an axonal marker, confirming the cellular localization previously described in literature [23, 24].

HuR–silencing increased spinal myelin content in SNI mice

The level of myelination within the central nervous system (CNS) was detected in spinal cord samples of SNI. No significant difference in myelin basic protein (MBP) expression between ipsilateral and contralateral side of spinal cord tissue of dODN–treated SNI mice was observed. I.n. anti–HuR ASO treatment increased the levels of MBP, while no effect was detected following anti–HuD ASO administration (Fig. 5A). These results were consistent with Luxol Fast Blue (LFB) staining of the spinal sections. Quantitative analysis showed a significant increased staining in anti–HuR ASO–treated SNI mice compared to dODN mice (Fig. 5B), confirming a significant increase in the myelin content. Anti–HuD ASO mice did not show any modification in LFB staining compared to control. Representative images showed a white marking much more evident in the anti–HuR ASO group compared to the other groups (Fig. 5C).

The level of myelination within the peripheral nervous system was investigated in sciatic nerve samples of SNI mice. Myelination of Schwann cells was detected by quantifying the S100 protein levels and a significant decrease of the expression of this protein was detected in the ipsilateral side of the dODN–treated SNI mice. Both i.n. anti–HuR and anti–HuD ASO groups did not alter S100 expression (Fig. 5D). Representative images of sciatic nerves showed the different intensity of S100 immunostaining between contra and ipsilateral side of control mice and the lack of effect by ASO treatments (Fig. 5E).

Interplay between HuR and HuD in the promotion of spinal expression of MBP

To evaluate the presence of a connection between HuR and HuD, we investigated if HuR silencing might lead to a modification in the spinal HuD protein levels in conditions of neuropathic pain associated with the EAE models. While HuR was upregulated in both models (Fig. 6A), HuD showed a different pattern of expression, being unaltered in PLP–EAE and drastically downregulated in MOG–EAE mice (Fig. 6B). Anti–HuR ASO downregulated HuR expression (Fig. 6A) while it increased HuD expression in the PLP–EAE and MOG–EAE models (Fig. 6B). HuD protein levels were further reduced by treatment with anti–HuD ASO (Fig. 6B).

Anti–HuR ASO also influenced MBP expression with a different pattern in the models investigated showing a prominent increase in the MOG–EAE mice (Fig. 6C), the model in which HuD was more drastically upregulated.

To support the hypothesis of a neuroprotective effect of the anti–HuR ASO mediated by HuD overexpression, immunofluorescence micrographs confirmed the GAP43 (red) and HuD (green) reduced expression in MOG–EAE mice and their overexpression following anti–HuR ASO treatment. Merge images showed an abundant localization of HuD in GAP43 expressing cells (Figure 6C), as confirmed by quantification analysis (anti–HuR ASO Mander’s Coefficient 0.89±0.03).

Neuropathic pain represents the most difficult to treat pain condition in MS patients. The unmet need for effective treatments has led to increasing attempts to define the complex pathophysiology of neuropathic pain. Recent evidence related neuropathic pain to demyelination, a condition which is usually related to an unresolved neuroinflammation. Thus, targeting demyelination appears a promising disease–modifying strategy to address neuropathic pain progression rather than merely suppress symptoms. In the present study we investigated HuR and HuD, RNA–binding proteins (RBP) belonging to the ELAV family, and we observed that restoration of an aberrant expression of HuR in mouse models of MS counteracted demyelination and neurodegeneration and attenuated clinical symptoms.

Spinal cord sections of MOG–EAE mice (model of chronic MS) showed a marked demyelination that was largely improved by i.n. anti–HuR ASO treatment. These results are consistent with previous findings on PLP–EAE mice (model of relapsing–remitting MS) treated intrathecally with an anti–HuR ASO [37]. I.n. anti–HuR ASO ameliorated mechanical allodynia and attenuated motor disability in both PLP–EAE and MOG–EAE mice. MS–associated neuropathic pain develops as a direct or indirect result of demyelinating lesions in the brain and spinal cord in areas involved in pain perception [43, 44] highlighting a functional relation of spinal HuR overexpression to neuropathic pain and spinal myelination. Indeed, the positive effects on demyelination temporally coincided with the attenuation of mechanical allodynia and motor disability, suggesting that the control of neuropathic pain might underly a protective activity towards demyelination.

Myelin restoration can be achieved by improving myelination as well as by dampening pro–demyelinating processes mainly consisting of aberrant immune and inflammatory responses. Considering that in MS patients inflammation declines with disease duration while neurodegeneration proceeds and activated microglia persists in all lesions in progressive MS, using the MOG–EAE model there was the need to answer the question if anti–HuR ASO effects resulted from a direct mechanism on myelination or indirectly through a central immune/inflammatory suppression. ASO reduced the BBB leakage in the brain and spinal cord during MOG–EAE in the absence of any significant reduction of circulating pro–inflammatory cytokines. Consistent with our findings, a recent clinical study showed a reduced HuR level on peripheral blood mononuclear cells from MS patients compared with healthy subject [45]. By comparing MOG–EAE and PLP–EAE mice, spinal activated microglia were more abundant and more intensively reduced by anti–HuR ASO in the relapsing–remitting model. Conversely, the chronic model showed a lower degree of microglia activation that remained almost unmodified by ASO treatment. These data are consistent with the clinical observation of an inflammation decline from the relapsing–remitting MS to the progressive MS and let hypothesize that an immune/inflammatory suppression, while relevant for the PLP–EAE model, is not prominently involved in the ASO–induced remyelinating and pain–relieving activity in the chronic model.

HuR is ubiquitously expressed and it has been detected in activated microglia as well as in neurons in the spinal cord of EAE mice [37]. Thus, the protective effect towards demyelination in MOG–EAE mice can be produced, at least in part, through a neuron–mediated mechanism. An interplay between HuR and nELAV RBPs in neuronal differentiation in vitro has been demonstrated, by which the appearance of nELAV in neuroblasts downregulates HuR expression [21]. HuD, the best studied member of the nELAVs, has showed a role in promoting neuroprotection in neuropathies. HuD–dependent GAP–43 [24, 46] and BDNF [47] increase has been shown in the DRG and axons during nerve regeneration in peripheral neuropathy models and growing evidence associates mRNAs HuD target to neurological and neurodegenerative disorders [48–50]. Thus, we investigated the presence of a connection between HuR and HuD. While both models showed spinal HuR overexpression, they showed a different pattern of HuD expression. MOG mice showed a drastic loss of HuD protein in the spinal cord that temporally coincided with HuR overexpression. These events were also concomitant to myelin loss and decreased expression of axonal (Neurofilament H) and neuroregeneration (GAP43) markers. HuR silencing restored to basal levels HuD protein as well as Neurofilament H and GAP43. Dampening axonal degeneration could extend the period during which axons could be remyelinated, thus favoring myelin restoration. Since GAP43 [24] and Neurofilament H [51] are among the main targets of HuD, these findings let hypothesize that the neuroprotective activity produced by anti–HuR ASO in chronic EAE could underlie an indirect mechanism related to restoration of HuD expression. The two–week period of repeated treatment needed to ease pain hypersensitivity in MOG-EAE mice further support the hypothesis of a long–lasting indirect mechanism.

PLP-EAE mice showed unaltered HuD, GAP43 and Neurofilament H levels that remained almost unmodified by anti–HuR ASO treatment, indicating that in this model the HuD–mediated spinal neuroprotection does not appear prominently activated. PLP–EAE is a model that mirrors the early stages of MS in which neuroinflammation is a main feature. Previous results showed that intrathecal HuR silencing in PLP-EAE mice induced myelin restoration related to the attenuation of microglia–mediated neuroinflammation as well as a pain–relieving activity that appeared starting from one week of treatment and persisted up to the end of experimentation [37]. To better define the role of HuR to demyelination–associated neuropathic pain, the effect of i.n. anti–HuR ASO on spinal myelination was investigated in SNI mice, a model of peripheral mononeuropathy. Similarly to EAE mice, SNI mice showed an overexpression of spinal HuR with a prominent microglial localization suggesting HuR upregulation as a common trait for these neuropathic pain conditions. Anti-HuR ASO treatment inhibited microglia-mediated spinal neuroinflammation and attenuated the nociceptive phenotype [23]. Even if demyelination was not detected in spinal cord sections, HuR knockdown largely increased in the spinal myelin content. In mice with a sciatic nerve lesion, neuropathic pain is sustained by the recruitment of monocytes/macrophages at peripheral level and by a microglia–mediated spinal pro–inflammatory response [52, 53]. Thus, consistent with the role of HuR in promoting a spinal neuroinflammatory response through microglia activation described in humans and animal models [17, 23], the attenuation of microglia–mediated neuroinflammation appears as a plausible mechanism for the positive effect on spinal myelination by HuR silencing.

These findings highlight a multitarget activity of HuR that allows the anti-HuR ASO to control neuroinflammation, through microglia mediated mechanisms, and to attenuate neurodegeneration, through neuronal mechanisms mediated by the overexpression of HuD. Thus, beside the microglia–mediated anti–neuroinflammatory activity, the activation of HuD–mediated pathways represents an additional mechanism for myelin protection that is promoted when neurodegeneration represents a prominent event. Anyway, both mechanisms are related to myelin protection making anti–HuR ASO an innovative approach to attenuate demyelination–associated neuropathic pain in early and late disease states.

A further improvement for the neuropathic pain therapy is represented by the i.n. delivery route. Even though the clinical efficacy of ASO–based therapies has been assessed, the difficulty to deliver ODNs to the CNS because of their poor capability to cross the BBB limits their application in pathological conditions that require CNS penetration. The i.n. drug delivery represents a simple, noninvasive, allowing self–medication in patients, route of administration that facilitates a rapid achievement of therapeutic drug concentrations in the brain and cerebrospinal fluid. This way could help avoid unpleasant delivery methods (i.e. intrathecal) currently required for ASO–based therapies. The wide interest aroused by this delivery route is demonstrated by 378 clinical trials under way in the United States alone using i.n. administration [54].

In conclusion, spinal HuR overexpression was observed in both chronic and relapsing remitting EAE mice and i.n. administration of an anti–HuR ASO attenuated pain hypersensitivity. This effect was induced by promoting myelin restoration through either HuD–mediated neuroprotective phenomena in chronic EAE mice or by dampening pro–inflammatory response mediated by spinal microglia activation in relapsing–remitting EAE mice. Present findings could represent an important step to better understand the pathophysiological role of post–transcriptional regulation of gene expression in demyelination-associated neuropathic pain conditions and make targeting HuR an innovative and promising step toward disease–modifying strategies for the management of neuropathic pain states.

Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests: The authors have no relevant financial or non-financial interests to disclose.

Author contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Vittoria Borgonetti. The first draft of the manuscript was written by Nicoletta Galeotti and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval: The experimental protocol was carried out after approval by the Animal Care and Research Ethics Committee of the University of Florence, Italy, under license from the Italian Department of Health (641/2017–PR) and in compliance with international laws and policies (Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes; Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). All studies involving animals are reported in accordance with the ARRIVE 2.0 guidelines for experiments involving animals [55]. All effort was taken to minimize the number of animals used and their suffering.

Consent to participate: Not applicable.

Consent to publish: Not applicable.

Acknowledgment: Not applicable.

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Myelin restoration by antisense modulation of HuR and HuD ELAV RNA binding protein expression in mouse models of multiple sclerosis

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